Is there concordance in attitudes and beliefs between parents and scientists about autism spectrum disorder?

There is no reported investigation comparing concordance in attitudes and beliefs about autism spectrum disorder between parents of children with autism spectrum disorder and scientists who research autism spectrum disorder. To investigate the level of concordance between these groups on causes of autism, priorities of research, perceived stigma, and disclosure of genetic test results, telephone interviews were conducted. Parents (n = 502) were recruited from the Simons Simplex Collection, and research scientists (n = 60) were recruited from investigators funded by the Simons Foundation. Response rates were notable (parents 91%, scientists 80%). Parents and scientists differed significantly regarding beliefs of the likely major cause of autism (p = 0.007) and priorities for further research (p < 0.001). Scientists believed in genetic causes while many parents believed in vaccines as the cause of autism. Parents (37%) were more likely to hesitate vaccinating their child (p < 0.001). In contrast, there was strong concordance regarding extent of perceived stigma (95% vs 92%) and preferences for disclosure of genetic test results, including incidental findings. While scientists believed communication important, paradoxically fewer than half reported it important for scientists to communicate directly with parents. Better communication between parents and scientists should improve mutual understanding and ultimately the health and well-being of children with autism spectrum disorder and their families.

The pattern of cortical dysfunction in a mouse model of a schizophrenia-related microdeletion.

We used a mouse model of the schizophrenia-predisposing 22q11.2 microdeletion to evaluate how this genetic lesion affects cortical neural circuits at the synaptic, cellular, and molecular levels. Guided by cognitive deficits, we demonstrated that mutant mice display robust deficits in high-frequency synaptic transmission and short-term plasticity (synaptic depression and potentiation), as well as alterations in long-term plasticity and dendritic spine stability. Apart from previously reported reduction in dendritic complexity of layer 5 pyramidal neurons, altered synaptic plasticity occurs in the context of relatively circumscribed and often subtle cytoarchitectural changes in neuronal density and inhibitory neuron numbers. We confirmed the pronounced DiGeorge critical region 8 (Dgcr8)-dependent deficits in primary micro-RNA processing and identified additional changes in gene expression and RNA splicing that may underlie the effects of this mutation. Reduction in Dgcr8 levels appears to be a major driver of altered short-term synaptic plasticity in prefrontal cortex and working memory but not of long-term plasticity and cytoarchitecture. Our findings inform the cortical synaptic and neuronal mechanisms of working memory impairment in the context of psychiatric disorders. They also provide insight into the link between micro-RNA dysregulation and genetic liability to schizophrenia and cognitive dysfunction.

Deficiency of Dgcr8, a gene disrupted by the 22q11.2 microdeletion, results in altered short-term plasticity in the prefrontal cortex.

Individuals with 22q11.2 microdeletions have cognitive and behavioral impairments and the highest known genetic risk for developing schizophrenia. One gene disrupted by the 22q11.2 microdeletion is DGCR8, a component of the "microprocessor" complex that is essential for microRNA production, resulting in abnormal processing of specific brain miRNAs and working memory deficits. Here, we determine the effect of Dgcr8 deficiency on the structure and function of cortical circuits by assessing their laminar organization, as well as the neuronal morphology, and intrinsic and synaptic properties of layer 5 pyramidal neurons in the prefrontal cortex of Dgcr8(+/-) mutant mice. We found that heterozygous Dgcr8 mutant mice have slightly fewer cortical layer 2/4 neurons and that the basal dendrites of layer 5 pyramidal neurons have slightly smaller spines. In addition to the modest structural changes, field potential and whole-cell electrophysiological recordings performed in layer 5 of the prefrontal cortex revealed greater short-term synaptic depression during brief stimulation trains applied at 50 Hz to superficial cortical layers. This finding was accompanied by a decrease in the initial phase of synaptic potentiation. Our results identify altered short-term plasticity as a neural substrate underlying the cognitive dysfunction and the increased risk for schizophrenia associated with the 22q11.2 microdeletions.

The Simons Simplex Collection: a resource for identification of autism genetic risk factors.

In an effort to identify de novo genetic variants that contribute to the overall risk of autism, the Simons Foundation Autism Research Initiative (SFARI) has gathered a unique sample called the Simons Simplex Collection (SSC). More than 2000 families have been evaluated to date. On average, probands in the current sample exhibit moderate to severe autistic symptoms with relatively little intellectual disability. An interactive database has been created to facilitate correlations between clinical, genetic, and neurobiological data.

Rescuing Z+ agrin splicing in Nova null mice restores synapse formation and unmasks a physiologic defect in motor neuron firing.

Synapse formation at the neuromuscular junction (NMJ) requires an alternatively spliced variant of agrin (Z(+) agrin) that is produced only by neurons. Here, we show that Nova1 and Nova2, neuron-specific splicing factors identified as targets in autoimmune motor disease, are essential regulators of Z(+) agrin. Nova1/Nova2 double knockout mice are paralyzed and fail to cluster AChRs at the NMJ, and breeding them with transgenic mice constitutively expressing Z(+) agrin in motor neurons rescued AChR clustering. Surprisingly, however, these rescued mice remained paralyzed, while electrophysiologic studies demonstrated that the motor axon and synapse were functional-spontaneous and evoked recordings revealed synaptic transmission and muscle contraction. These results point to a proximal defect in motor neuron firing in the absence of Nova and reveal a previously unsuspected role for RNA regulation in the physiologic activation of motor neurons.

NRG1 and synaptic function in the CNS.

Recent genetic evidence indicates that neuregulin 1 (NRG1) and its receptor erbB4 may be susceptibility genes in schizophrenia, but their function in CNS synaptic transmission and circuitry is not well understood. In this issue of Neuron, studies from Li et al. and Woo et al. show that NRG1 and erbB4 regulate transmission at brain glutamate and GABA synapses. These findings raise the possibility of synaptic defects in schizophrenia.

An acute effect of neuregulin 1 beta to suppress alpha 7-containing nicotinic acetylcholine receptors in hippocampal interneurons.

We examined rapid effects of neuregulin (NRG) on nicotinic acetylcholine (ACh) receptors in interneurons located in the stratum radiatum of the hippocampus. Two types of response were detected by whole-cell recordings after brief pulses of ACh. One type was a rapidly rising and falling (monophasic) current that was blocked by methyllycaconitine. The other type was a similar fast response followed by a more slowly rising and falling current. The slow component of the biphasic response was resistant to methyllycaconitine. Perfusion or local application with NRG 1beta rapidly decreased fast inward ACh currents. NRG 1beta had no effect on slow responses. NRG 1beta suppression was abolished by the ErbB tyrosine kinase inhibitor PD 158780 (4-[(3-bromophenyl) amino]-6-(methylamino)-pyrido[3,4-d]pyridimine). The NRG 1beta effect was also inhibited by phalloidin and cytochalasin D. Furthermore, NRG 1beta decreased the number of surface Alexa Fluor 488 alpha-bungarotoxin binding sites. We believe that the NRG 1beta-induced inhibition of ACh currents is because of receptor internalization trigged by protein tyrosine phosphorylation. Significantly, fast nicotinic EPSCs evoked in the presence of muscarinic, ionotropic glutamate, and GABA receptors antagonists were also reduced by NRG 1beta. Thus, short-term as well as long-term effects of NRG must be taken into consideration in studies of ACh receptor-mediated synaptic efficacy in the CNS.

Neuregulin effect on quantal content dissociated from effect on miniature endplate potential amplitude.

Members of the neuregulin family of signaling proteins increase transcription of acetylcholine receptor (AChR) subunit genes in muscle fibers and the number of AChRs in the muscle membrane. In adult mice heterozygous for targeted deletion of type I neuregulins (Ig-NRG(+/-)), postsynaptic AChR density was decreased and transmitter release was increased. We examined the relationship between functional AChR density and ACh release in postnatal day 7 (P7), P14, and adult NRG-deficient mice. Here we report that changes in postsynaptic sensitivity and transmitter release are not temporally coupled during postnatal development in Ig-NRG-deficient mice. Although miniature endplate potential (MEPP) amplitude was decreased compared with control in P7 Ig-NRG(+/-) mice, quantum content was not increased. Quantum content was increased in adult heterozygotes despite normal MEPP amplitudes. Thus, during postnatal maturation, both quantal size and quantum content were influenced by decreased Ig-NRG expression, although the effects were dissociated in time.

Neuregulin-1 increases the proliferation of neuronal progenitors from embryonic neural stem cells.

Neuregulins are a family of proteins expressed in the developing brain and in brain regions that continue to undergo neurogenesis in adult animals. We investigated the effects of neuregulins on embryonic neural stem cells (NSCs) isolated from E11 mouse telencephalon. Treatment of basic fibroblast growth factor (bFGF)-expanded neurosphere cultures with the EGF-like domain of neuregulin1-beta1 (NRG-1(177-244)) resulted in a 4-fold increase of bromodeoxyuridine (BrDU)-labeled cells, suggesting that NRG-1 stimulated proliferation. The majority of the BrdU-positive cells co-labeled with an antibody against MAP2, indicating that the proliferating cells were neuronal. No BrDU labeling was seen in GFAP- or O4-positive cells. In NRG-1-treated cultures, many of the MAP2-positive cells co-labeled with an anti-nestin antibody, suggesting that these cells are neuron-restricted progenitors (NRPs). Few MAP2/nestin-positive cells were seen in control cultures. The increase in the number of neuronal cells in NRG-1-treated cultures was due to increased proliferation of MAP2-positive cells rather than the regulation of cell survival or fate determination. These results suggest that neuregulins are mitogenic to NRPs, thus endogenous neuregulins may play important roles during CNS neurogenesis.

Stem cells: science, policy, and ethics.

Human embryonic stem cells offer the promise of a new regenerative medicine in which damaged adult cells can be replaced with new cells. Research is needed to determine the most viable stem cell lines and reliable ways to promote the differentiation of pluripotent stem cells into specific cell types (neurons, muscle cells, etc). To create new cell lines, it is necessary to destroy preimplantation blastocysts. This has led to an intense debate that threatens to limit embryonic stem cell research. The profound ethical issues raised call for informed, dispassionate debate.

Neuregulin induces the expression of transcription factors and myosin heavy chains typical of muscle spindles in cultured human muscle.

Neuregulin (NRG) (also known as ARIA, GGF, and other names) is a heparin sulfate proteoglycan secreted into the neuromuscular junction by innervating motor and sensory neurons. An integral part of synapse formation, we have analyzed NRG-induced changes in gene expression over 48 h in primary human myotubes. We show that in addition to increasing the expression of acetylcholine receptors on the myotube surface, NRG treatment results in a transient increase of several members of the early growth response (Egr) family of transcription factors. Three Egrs, Egr1, -2, and -3, are induced within the first hour of NRG treatment, with Egr1 and -3 RNA levels showing the most significant increases of approximately 9- and 16-fold, respectively. Also noted was a corresponding increase in protein levels for both of these transcription factors. Previous literature indicates that Egr3 expression is required for the formation of muscle spindle fibers, sensory organs that are distinct from skeletal muscle contractile fibers. At the molecular level, muscle spindle fibers express a unique subset of myosin heavy chains. Two isoforms of the myosin heavy chain, the slow development and neonatal, were found to be increased in our myotube cultures after 48 h of treatment with NRG. Taken together, these results indicate that not only can NRG induce the expression of a transcription factor key to spindle fiber development (Egr3), but that a portion of this developmental process can be replicated in vitro.

Neuregulin-1 suppresses muscarinic receptor expression and acetylcholine-activated muscarinic K+ channels in cardiac myocytes.

The neuregulin-1 family of growth factors regulates nicotinic acetylcholine receptor synthesis in skeletal muscle, but its role in cardiac myogenesis remains unclear. Here, we investigate the involvement of neuregulins in the development of cardiac cholinergic responsiveness. Treatment of chick cardiac myocytes with neuregulin-1 inhibited mRNA expression of the M4 muscarinic receptor, but not the M2 receptor. In addition, mRNA levels of GIRK1 were reduced in myocytes by treatment with neuregulin-1. Activation of cholinergic receptors in cultured chick atrial myocytes by carbachol produced an outward potassium current (I(K(ACh))), which was attenuated by 24-48-h pre-treatment with neuregulin-1. These data suggest that neuregulins can regulate cardiac parasympathetic tone and may be involved in the pathogenesis of cardiac arrhythmias and heart failure.

Differentiation-dependent regulation of skeletal myogenesis by neuregulin-1.

Neuregulins comprise a group of growth factor proteins that regulate the differentiation of skeletal muscle. Here, we report that neuregulins are regulators of myogenic differentiation and stimulate mitogenesis in L6 skeletal myoblasts. The mitogenic response to neuregulin-1 was differentiation-dependent and observed only in aligned, differentiating cells. Treatment of these cells with neuregulin-1 increased [3H]thymidine incorporation and cell proliferation by 2- to 5-fold, while a minimal increase was seen in proliferating myoblasts. Neuregulin-1 did not induce DNA synthesis in fused, multinucleated myotubes. The increased DNA synthesis correlated with downregulation of myogenin and inhibition of myoblast fusion and myotube formation. These data suggest that neuregulins may regulate skeletal myogenesis in vivo and that this regulation is dependent on the state of differentiation of the myocytes.

Type 3 reovirus neuroinvasion after intramuscular inoculation: direct invasion of nerve terminals and age-dependent pathogenesis.

Neonatal but not adult mice are vulnerable to reovirus invasion of the central nervous system after peripheral inoculation. After hindlimb injection, type 3 reovirus travels via the sciatic nerve to replicate in spinal cord motor neurons before spread to the brain and development of lethal encephalitis. Here we provide ultrastructural evidence for direct reovirus invasion of unmyelinated neonatal motor nerve terminals within 2 h and replication in spinal cord motor neurons within 14 h after hindlimb injection of 1-day-old mice. In adult mice, resistance to reovirus lethality after intracranial (IC) injection correlates with the restriction of virus growth in cortical neurons. We found that neuroinvasion also is age dependent after intramuscular injection. Virus lethality and CNS infection decreased sharply during the first postnatal week, while lethality after IC injection continued for 2 additional weeks. Mice inoculated at 7 days of age with high virus doses suffered paralysis of the injected limb, but significant brain infection was not lethal. These results suggest that reovirus invasion of the neonatal CNS is restricted by several progressive age-dependent mechanisms.

Neuregulin expression at neuromuscular synapses is modulated by synaptic activity and neurotrophic factors.

The proper formation of neuromuscular synapses requires ongoing synaptic activity that is translated into complex structural changes to produce functional synapses. One mechanism by which activity could be converted into these structural changes is through the regulated expression of specific synaptic regulatory factors. Here we demonstrate that blocking synaptic activity with curare reduces synaptic neuregulin expression in a dose-dependent manner yet has little effect on synaptic agrin or a muscle-derived heparan sulfate proteoglycan. These changes are associated with a fourfold increase in number and a twofold reduction in average size of synaptic acetylcholine receptor clusters that appears to be caused by excessive axonal sprouting with the formation of new, smaller acetylcholine receptor clusters. Activity blockade also leads to threefold reductions in brain-derived neurotrophic factor and neurotrophin 3 expression in muscle without appreciably changing the expression of these same factors in spinal cord. Adding back these or other neurotrophic factors restores synaptic neuregulin expression and maintains normal end plate band architecture in the presence of activity blockade. The expression of neuregulin protein at synapses is independent of spinal cord and muscle neuregulin mRNA levels, suggesting that neuregulin accumulation at synapses is independent of transcription. These findings suggest a local, positive feedback loop between synaptic regulatory factors that translates activity into structural changes at neuromuscular synapses.